Currently, I'm using mT/mG reporter mice to check the cre activity of this Ert2 cre mice.
mT/mG mouse is supposed to autofluoresce without any additional staining or immunohistochemistry necessary. Nestin.CreERT2 is the transgenic mice that express CreERT2 under the control of the nestin promoter and enhancer. the cre will become activated by administration of tamoxifen.
After the cross breeding, my study is about neonatal brain,so the fist IP inject at P6 of pups, 0.25mg per body weight. 48h later, the second IP injection at P8, then 24h later, the P9 pups were perfused with cold 0.9% saline. After brain dissociation, brains were fixed 24h in PFA at 4, cryoprotected in 30% sucrose overnight at 4, and embedded in OCT for cryosection. 20 micrometer sections were mounted on slices directly.
the problem is I cannot see any fluorescence signal by using a traditional fluorescent microscope, even without any red fluorescence (mT, tdTomato), I'm thinking do I expose the slice in light a long time? Does this effect a lot? Is there some problems of the microscope? is it better change to confocal? Do I have some problems about the administration, such as the duration, the amount?
ANY SUGGESTIONS WILL BE APPRECIATED ?
I believe that 24 hours of fixation in PFA will kill the EGFP fluoresence. You can still try staining with an anti-GFP antibody. Otherwise you can section fresh frozen tissue and examine it before fixation.
Some reporter mice have low florescence levels. That could be solved as Gregory points using an anti-GFP antibody.
But you also might want to confirm that you are inducing CRE expression using a anti-CRE antibody.
In my hands EGFP signal is pretty resistant and I see some fluorescence even after an In Situ hybridization protocol (without protease treatment).
Thank you so much, guys, thank you for your elaborate advises, Hussein. this tamoxifen inducible transgenic mice is kind of tricky, especially in under P9 pups. Anyway, I will follow your suggestions and try the fresh frozen section next litter first. I want to avoid the fluorescence decrease as possible as I can. Later, depends on the results, the staining is a good choice.
Cheers, guys!
Hi Tao, if you have less GFP-stain and high aufluorescence I recommand IHC with Anti-GFP and detection with DAB and counterstain with Haematoxyline. If you are working wirh PFA fixed and cryprotected brain tissue you can use the floating methode. The advantage is the GFP-antibody can reach the epitopes from both sides of the section. Overnight reaction in the primary antibody at room temperature is recommanded. Good luck!
Hello Tao,
I don't know exactly which reporter line you are using, but are you sure that killing the animals 3 days after the first injection is enough time to have detectable expression of your reporter? For my experiments using inducible Cre lines I normally wait a week after injecting tamoxifen to have strong labelling.
Also, keep in mind that Nestin is only expressed in neural progenitor cells, and that depending on the timing of induction you will label different cells. Since you are injecting tamoxifen postnatally, when most of the neurogenesis is done, you will probably only label a small number of cells.
Look at Fig 1D of the following publication to have an idea of where you would expect to see labelled cells. Your samples should look be similar to what is shown in panel d4 (tamoxifen injected at P7).
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708938/
I hope this helps!
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